U.S. patent application number 13/817985 was filed with the patent office on 2013-10-24 for density-based separation of biological analytes using multiphase systems.
This patent application is currently assigned to President and Fellows of Harvard College a University. The applicant listed for this patent is Ashok A. Kumar, Charles R. Mace, George M. Whitesides, Dyann F. Wirth. Invention is credited to Ashok A. Kumar, Charles R. Mace, George M. Whitesides, Dyann F. Wirth.
Application Number | 20130280693 13/817985 |
Document ID | / |
Family ID | 44545953 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130280693 |
Kind Code |
A1 |
Mace; Charles R. ; et
al. |
October 24, 2013 |
DENSITY-BASED SEPARATION OF BIOLOGICAL ANALYTES USING MULTIPHASE
SYSTEMS
Abstract
The disclosed methods use a multi-phase system to separate
samples according to the density of an analyte of interest. The
method uses a multi-phase system that comprises two or more
phase-separated solutions and a phase component such as a
surfactant or polymer. The density of the analyte of interest
differs from the densities of the rest of the sample. The density
of the analyte of interest is substantially the same as one or more
phases. Thus, when the sample is introduced to the multi-phase
system, the analyte of interest migrates to the phase having the
same density as the analyte of interest, passing through one or
more phases sequentially.
Inventors: |
Mace; Charles R.; (Auburn,
NY) ; Kumar; Ashok A.; (Cambridge, MA) ;
Wirth; Dyann F.; (Boston, MA) ; Whitesides; George
M.; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mace; Charles R.
Kumar; Ashok A.
Wirth; Dyann F.
Whitesides; George M. |
Auburn
Cambridge
Boston
Newton |
NY
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
President and Fellows of Harvard
College a University
|
Family ID: |
44545953 |
Appl. No.: |
13/817985 |
Filed: |
August 22, 2011 |
PCT Filed: |
August 22, 2011 |
PCT NO: |
PCT/US2011/048678 |
371 Date: |
July 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61375532 |
Aug 20, 2010 |
|
|
|
Current U.S.
Class: |
435/2 ; 435/34;
435/5; 435/6.1; 435/7.24 |
Current CPC
Class: |
G01N 33/491 20130101;
B03B 5/28 20130101; G01N 33/18 20130101; B03D 3/00 20130101; G01N
33/5375 20130101; G01N 33/57492 20130101; B03B 5/442 20130101 |
Class at
Publication: |
435/2 ; 435/34;
435/5; 435/6.1; 435/7.24 |
International
Class: |
G01N 33/49 20060101
G01N033/49; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of analyzing or separating a sample comprising one or
more biological analytes of interest using a multi-phase system,
comprising: a) providing a multi-phase system comprising two or
more phase-separated solutions, wherein each phase comprises a
phase component selected from the group consisting of a polymer, a
surfactant and combinations thereof, wherein at least one phase
comprises a polymer; each said phase has an upper and a lower phase
boundary; and each of the two or more phases has a different
density and the phases, taken together, represent a density
gradient; and b) introducing a biological sample comprising one or
more biological analytes of interest without disrupting the
phase-separated solution; and c) allowing each of the biological
analytes to migrate to a location in the multi-phase system that is
characteristic of its density, wherein during migration the sample
contacts one or more of the two or more phases sequentially.
2. The method of claim 1, wherein the at least two phases share a
common solvent.
3. The method of claim 2 wherein the multi-phase system is an
aqueous system and the common solvent is an aqueous solvent.
4. The method of claim 1, wherein the multi-phase system is a
non-aqueous system and the common solvent is an organic
solvent.
5. The method of claim 1, wherein the phase components are selected
to be biologically compatible.
6. The method of claim 1, wherein the biological sample comprises
cells.
7. The method of claim 1, wherein the cells are selected from the
group consisting of animal, plant, protozoan, and prokaryotic
cells.
8. The method of claim 6, wherein one or more phases comprises a
lysing agent to cause the cells to lyse, the biological analyte of
interest being recovered from cell lysate.
9. The method of claim 1, wherein the biological analyte is
selected from the group consisting of organelles, cell fragments,
cell membranes, cell membrane fragments, viruses, virus-like
particles, bacteriophage, cytosolic proteins, secreted proteins,
signaling molecules, embedded proteins, nucleic acid/protein
complexes, organelles, minicells, nucleic acid precipitants,
chromosomes, nuclei, mitochondria, chloroplasts, flagella,
biominerals, protein complexes, and protein aggregates.
10. The method of claim 1, wherein the biological sample comprises
one or more parasites selected from the group consisting of worms,
insects, protozoa, arachnids, and arthropods.
11. The method of claim 1, wherein the biological sample comprises
a biological fluid.
12. The method of claim 1, wherein the biological sample is
selected from the group consisting of food, juice, and milk.
13. The method of claim 12, wherein the biological sample is tested
for contaminants selected from the group consisting of pathogens,
pests, heavy metals, and pesticides.
14. The method of claim 1, wherein the biological sample comprises
one or biological carriers selected from the group consisting of
whole blood, plasma, serum, sweat, feces, urine, saliva, tears,
vaginal fluid, prostatic fluid, gingival fluid, amniotic fluid,
intraocular fluid, cerebrospinal fluid, seminal fluid, sputum,
ascites fluid, pus, nasopharengal fluid, wound exudate fluid,
aqueous humour, vitreous humour, bile, cerumen, endolymph,
perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid,
sebum, vomit, and combinations thereof.
15. The method of claim 1, wherein the biological analyte is
separated and analyzed to distinguish cell states selected from the
group consisting of normal cells, diseased cells, parasitized
cells, cancer cells, foreign cells, and infected cells.
16. The method of claim 1, wherein the sample comprises a plurality
of analytes and each analyte migrates to a different location in
the phase-separated system.
17. The method of claim 1, wherein after migration, the analyte
resides at a boundary location.
18. The method of claim 1, wherein the boundary location is at an
interface between a phase with a density greater than the density
of the analyte and a phase with a density that is less than the
density of the analyte.
19. The method of claim 1, wherein after migration, the analyte
resides within a phase of the phase-separated system whose density
matches the density of the analyte.
20. The method of claim 1, wherein the analyte/phase-separated
system is centrifuged to accelerate migration of the analyte.
21. The method of claim 1, wherein the analyte migrates under
gravitational forces.
22. The method of claim 1, wherein the analyte migrates under
buoyancy forces.
23. The method of claim 1, wherein the phase separated system is
supported in a column or test tube.
24. The method of claim 1, wherein the phase separated system is
supported in a capillary tube, plastic test tube, falcon tube,
culture tube, well plates, cuvette, along a filament, or on a
sheet.
25. The method of claim 1, wherein the surfactant is selected from
the group consisting of polysorbate, CHAPS,
polyoxyethylene-polyoxypropylene,
1-O-Octyl-.beta.-D-glucopyranoside,
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,
2-(Perfluoroalkyl)ethyl methacrylate, N,N-dimethyldodecylamine
N-oxide, polyethylene glycol dodecyl ether, sodium dodecyl sulfate,
sodium cholate, nonylphenol polyoxyethylene, benzylalkonium
chloride, and dodecyltrimethylammonium chloride.
26. The method of claim 1, wherein the polymer is selected from the
group consisting of dextran, polysucrose, poly(vinyl alcohol),
poly(2-ethyl-2-oxazoline), poly(methacrylic acid), poly(ethylene
glycol), polyacrylamide, polyethyleneimine, hydroxyethyl cellulose,
polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylic acid),
poly(2-acrylamido-2-methyl-1-propanesulfonic acid),
poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic
acid), polyallylamine, alginic acid, dextran sulfate, chondroitin
sulfate A, diethylaminoethyl-dextran,
poly(2-vinylpyridine-N-oxide), polydimethylsiloxane, and
poly(propylene glycol).
27. The method of claim 1, wherein the polymer or surfactant is
selected from the group consisting CHAPS,
polyoxyethylene-polyoxypropylene, dextran, polysucrose, poly(vinyl
alcohol), poly(2-ethyl-2-oxazoline), poly(ethylene glycol),
polyacrylamide, hydroxyethyl cellulose, polyvinylpyrrolidone,
carboxy-polyacrylamide, dextran sulfate, and
poly(2-vinylpyridine-N-oxide).
28. The method of claim 1, wherein the polymer is selected from the
group of GRAS polymers.
29. The method of claim 1, wherein the polymer is selected from the
group of homopolymers, random copolymers, block copolymers, graft
copolymers, ter-polymers, dendrimers, star polymers and
combinations thereof.
30. The method of claim 29, wherein the polymer is linear, branched
and/or cross-linked.
31. The method of claim 1, further comprising one or more additives
selected from the group consisting of miscible surfactants, salts,
dyes, nutrients, vitamins, antibiotics, anticoagulants, and
buffers.
32. A method of analyzing a sample comprising at least one analyte
of interest using a multi-phase system, comprising: a) providing a
sample comprising at least one analyte of interest; b) providing a
multi-phase system comprising two or more phases with clear
boundaries, wherein at least one of the phases comprises a phase
component, wherein the phase component is selected from the group
consisting of a polymer, a surfactant, and combinations thereof;
each of the two or more phases has a different density and the two
or more phases, taken together, represent a density gradient; and
the phases are phase-separated from each other; and c) introducing
the sample comprising a mixture of a tag molecule and the analyte
to the multi-phase system, wherein the analyte and the tag molecule
form a tag molecule-analyte adduct; and d) allowing the tag
molecule-analyte adduct to migrate to a location in the multiphase
system that is characteristic of its density, wherein during
migration the sample contacts one or more of the two or more phases
sequentially and the analyte and the tag molecule-analyte adduct
occupy different locations.
33. The method of claim 32, wherein the tag molecule-analyte adduct
is formed before or after the sample is introduced to the
multi-phase system.
34. A method of claim 32, wherein the sample comprises one or more
biological analytes of interest.
35. A method of claim 32, wherein the sample comprises one or more
non-biological analytes of interest.
36. A method of claim 32, wherein the multi-phase system comprises
one or more biologically compatible phases.
37. A method of claim 32, wherein the sample comprises the analyte
of interest and one or more impurities, the impurity having the
same density of the analyte, and the impurity having a different
density than the tag molecule-analyte adduct.
38. A method of claim 32, wherein the analyte of interest has an
affinity for the tag molecule, and wherein the analyte of interest
and tag molecule preferentially link to form a tag molecule-analyte
adduct, the tag molecule-analyte adduct being linked by a method
selected from the group consisting of covalent bonding,
non-covalent bonding, hybridization, complexation, electrostatic
interactions, and conjugation.
39. A method of claim 32, wherein the tag in the tag
molecule-analyte adduct has an affinity for one or more phase
components, the tag of the tag molecule-analyte adduct and the
phase component preferentially linking such that the tag
molecule-analyte adduct preferentially aggregates in one or more
phases containing phase components.
40. A method of claim 32, wherein the analyte of interest is a cell
selected from the group consisting of animal, plant, protozoan, and
prokaryotic cells.
41. A method of claim 32, wherein one or more phases cause the
cells to lyse, and the biological analyte of interest being
recovered from cell lysate.
42. A method of claim 32, wherein the sample comprises one or more
parasites selected from the group consisting of worms, insects,
protozoa, arachnids, and arthropods.
43. A method of claim 32, wherein the sample comprises one or
biological carriers selected from the group consisting of whole
blood, plasma, serum, sweat, feces, urine, saliva, tears, vaginal
fluid, prostatic fluid, gingival fluid, amniotic fluid, intraocular
fluid, cerebrospinal fluid, seminal fluid, sputum, ascites fluid,
pus, nasopharengal fluid, wound exudate fluid, aqueous humour,
vitreous humour, bile, cerumen, endolymph, perilymph, gastric
juice, mucus, peritoneal fluid, pleural fluid, sebum, vomit, and
combinations thereof.
44. A method according to any one of claims 40-43, wherein the
analyte of interest is separated and analyzed to distinguish cell
states selected from the group consisting of normal cells, diseased
cells, parasitized cells, cancer cells, foreign cells, and infected
cells.
45. A method of claim 32, wherein the surfactant is selected from
the group consisting of polysorbate, CHAPS,
polyoxyethylene-polyoxypropylene,
1-O-Octyl-.beta.-D-glucopyranoside,
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,
2-(Perfluoroalkyl)ethyl methacrylate, N,N-dimethyldodecylamine
N-oxide, polyethylene glycol dodecyl ether, sodium dodecyl sulfate,
sodium cholate, nonylphenol polyoxyethylene, benzylalkonium
chloride, and dodecyltrimethylammonium chloride.
46. A method of claim 32, wherein the polymer is selected from the
group consisting of dextran, polysucrose, poly(vinyl alcohol),
poly(2-ethyl-2-oxazoline), poly(methacrylic acid), poly(ethylene
glycol), polyacrylamide, polyethyleneimine, hydroxyethyl cellulose,
polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylic acid),
poly(2-acrylamido-2-methyl-1-propanesulfonic acid),
poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic
acid), polyallylamine, alginic acid, dextran sulfate, chondroitin
sulfate A, diethylaminoethyl-dextran,
poly(2-vinylpyridine-N-oxide), polydimethylsiloxane, and
poly(propylene glycol)
47. A method of claim 32, wherein the polymer or surfactant is
selected from the group consisting CHAPS,
polyoxyethylene-polyoxypropylene, dextran, polysucrose, poly(vinyl
alcohol), poly(2-ethyl-2-oxazoline), poly(ethylene glycol),
polyacrylamide, hydroxyethyl cellulose, polyvinylpyrrolidone,
carboxy-polyacrylamide, dextran sulfate, and
poly(2-vinylpyridine-N-oxide).
48. The method of claim 46, wherein the polymer is a type of
polymer selected from the group consisting of a homopolymer, random
copolymer, copolymer, terpolymer, block copolymer, linear polymer,
branched polymer, random polymer, crosslinked polymer, and
dendrimer system.
49. A kit for separating a sample comprising one or more biological
analytes of interest using a multi-phase system comprising: a) two
or more phase components selected from the group consisting of a
polymer, a surfactant, and combinations thereof; b) optionally a
tag molecule capable of binding the one or more biological analytes
of interest, wherein the tag molecule has a different density than
the biological analyte of interest; and c) instructions for: (i)
combining the two or more phase-separated solutions with a common
solvent to create a multi-phase system; (ii) optionally, combining
the biological analyte of interest and tag molecule, and (iii)
separating the biological analyte of interest from the sample.
50. The kit of claim 49, further comprising an aliquot of a common
solvent which, when combined with the two or more phase components,
provides a multiphase system.
51. The kit of claim 49, wherein the instructions direct that the
biological analyte of interest be combined with the tag molecule to
form a tag molecule-analyte adduct before introduction to the
multi-phase system.
52. The kit of claim 49, wherein the instructions further direct
that the biological analyte of interest and tag molecule be added
to the multi-phase system to combine to form a tag molecule-analyte
adduct in the multi-phase system.
53. The kit of claim 49, wherein the kit further comprises one or
more additives selected from the group consisting of miscible
surfactants, salts, dyes, nutrients, vitamins, antibiotics,
anticoagulants, and buffers for combination with the phase
components.
54. The kit of claim 49, wherein the multi-phase system comprises
one or more biologically compatible phases.
55. The kit of claim 49, wherein the kit comprises a tag that has
an affinity for one or more analytes of interest.
56. The kit of claim 49, wherein the kit comprises a tag that has
an affinity for one or more phase components.
57. The kit of claim 49, further comprising a lytic agent for
introduction into one or more phases of the multiphase system.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/375,532, filed on Aug. 20, 2010, which is hereby
incorporated by reference in its entirety. This application is also
related to the following applications, filed concurrently herewith,
the entire contents of which are incorporated herein by
reference:
[0002] PCT Patent Application No. ______, filed on Aug. 22, 2011,
entitled "MULTIPHASE SYSTEMS AND USES THEREOF," identified by
attorney docket number 0042697.251WO1;
[0003] PCT Patent Application No. ______, filed on Aug. 22, 2011,
entitled "MULTIPHASE SYSTEMS HAVING MULTIPLE PHASE PROPERTIES,"
identified by attorney docket number 0042697.251WO3; and
[0004] PCT Patent Application No. ______, filed on Aug. 19, 2011,
entitled "MULTIPHASE SYSTEMS FOR ANALYSIS OF SOLID MATERIALS,"
identified by attorney docket number 0042697.251WO4.
INCORPORATION BY REFERENCE
[0005] All non-patent literature, patents, patent applications and
publications cited herein are hereby incorporated by reference in
their entirety in order to more fully describe the state of the art
as known to those skilled therein as of the date of the invention
described herein.
BACKGROUND
[0006] It is known that the aqueous mixtures of two polymers such
as poly(ethylene glycol) (PEG) and dextran can separate
spontaneously into two aqueous phases, called aqueous two-phase
systems. Phase separation in aqueous solutions of polymers is an
extraordinary and underexplored phenomenon. When two aqueous
solutions of polymers are mixed, the resulting system is not
homogeneous; rather, two discrete phases, or layers, form. These
layers are ordered according to density and arise from the limited
interaction of the polymers for one another. In these systems, each
phase predominantly consists of water (upwards of 70-90% (w/v)),
while the polymer component is present in concentrations ranging
from micromolar to millimolar. A low interfacial tension and rapid
mass transfer of water-soluble molecules across the boundary
characterize the interface between layers.
[0007] Previous studies of partitioning between aqueous phases have
been limited to biphasic systems of immiscible polymers or
inorganic salts. These Aqueous Two-Phase Systems ("ATPS") are
exemplified by the poly(ethylene glycol)-dextran, dextran-Ficoll
systems, and a poly(ethylene glycol) system comprising
(NH.sub.4).sub.2SO.sub.4. Uses of these systems have focused on
applications in protein chemistry, cell partitioning, and
manufacturing.
[0008] Further, previous methods for separating and partitioning
components have been limited to, e.g., filtration, crystallization,
distillation, chromatography, and separation by hand. Many of these
methods have been proven difficult, imprecise, slow, expensive, and
unsuitable for use with diverse sample types and sizes, or
otherwise undesirable.
[0009] There is a need for simple, precise methods for separating
biological and non-biological samples based on density with
multi-phase systems suitable for use with diverse sample types and
sizes.
SUMMARY
[0010] Described herein are methods of separating or analyzing
analytes of interest using multi-phase systems ("MPS") comprising
two or more phases having different densities. In some embodiments,
MPS as described herein are used to separate analytes from each
other or from impurities and other objects in the sample when the
analytes migrate to phases characteristic of their densities, and
in so doing, contact each phase of the multi-phase system
sequentially. In some embodiments, a multi-phase system comprising
a phase component is used and the analyte contacts each phase of
the multi-phase system sequentially. As used herein, "sequential
contact" means that the analyte contacts and interacts with only
one phase (and its phase component) at a time except at the
interface where the analyte may contact and interact with two
adjacent phases simultaneously. That is, the interaction of the
analyte with the MPS occurs when the MPS has already phase
separated and not during the process of phase separation.
[0011] The multi-phase systems used in the methods disclosed herein
comprise two or more phases that are phase-separated from each
other, wherein each of the two or more phases comprises a phase
component. The phase component is one or more selected from the
group consisting of polymer, surfactant, or combinations thereof,
wherein at least one of the phase components is a polymer. The
phases in the multi-phase system can be aqueous or organic. In some
embodiments, at least one phase of the multi-phase system is
aqueous and at least one phase of the multi-phase system is
organic.
[0012] The phase component is selected from the group consisting of
a polymer, a surfactant and combinations thereof. The phase
"combination" refers to the combination of a polymer and a
surfactant, a combination of two or more polymers, a combination of
two or more surfactants, or a combination of any number of polymers
and any number of surfactants.
[0013] As used herein, MPS refers to a multi-phase system. When two
or more solutions containing a phase component are mixed, the
resulting system is not homogeneous; rather, two or more discrete
phases, or layers, form. These layers are ordered according to
density and arise from the exhibit limited interaction of the phase
components with one another. The two or more phases or solutions,
which exhibit limited interaction and form distinct phase
boundaries between adjacent phases. Each phase can be aqueous or
non-aqueous. The non-aqueous phase comprises an organic liquid or
an organic solvent.
[0014] As used herein, AMPS refers to an aqueous multi-phase
polymer system. ATPS refers to an aqueous two-phase polymer
system.
[0015] As used herein, an aqueous multi-phase polymer system
comprises two or more polymer aqueous solutions or phases, which
are phase-separated and in which at least two aqueous solutions
each comprises a polymer. In some embodiments, the aqueous
multi-phase polymer system can be combined with one or more
immiscible organic phases to form a multi-phase system.
[0016] As used herein, the use of the phrase "polymer" includes,
but is not limited to, the homopolymer, copolymer, terpolymer,
random copolymer, and block copolymer. Block copolymers include,
but are not limited to, block, graft, dendrimer, and star polymers.
As used herein, copolymer refers to a polymer derived from two
monomeric species; similarly, a terpolymer refers to a polymer
derived from three monomeric species. The polymer also includes
various morphologies, including, but not limited to, linear
polymer, branched polymer, random polymer, crosslinked polymer, and
dendrimer systems. As an example, polyacrylamide polymer refers to
any polymer including polyacrylamide, e.g., a homopolymer,
copolymer, terpolymer, random copolymer, block copolymer or
terpolymer of polyacrylamide. Polyacrylamide can be a linear
polymer, branched polymer, random polymer, crosslinked polymer, or
a dendrimer of polyacrylamide.
[0017] In one embodiment, the a method of analyzing or separating a
sample includes one or more biological analytes of interest using a
multi-phase system, comprising: a) providing a multi-phase system
comprising two or more phase-separated solutions, wherein each
phase comprises a phase component selected from the group
consisting of a polymer, a surfactant and combinations thereof,
wherein at least one phase comprises a polymer; each said phase has
an upper and a lower phase boundary; and each of the two or more
phases has a different density and the phases, taken together,
represent a density gradient; and b) introducing a biological
sample comprising one or more biological analytes of interest
without disrupting the phase-separated solution; and c) allowing
each of the biological analytes to migrate to a location in the
multi-phase system that is characteristic of its density, wherein
during migration the sample contacts one or more of the two or more
phases sequentially.
[0018] In another embodiment, a method of analyzing a sample
comprising at least one analyte of interest according to the
density of a tagged analyte of interest in a MPS is disclosed. The
method comprises: a) providing a sample comprising at least one
analyte of interest; b) combining the sample with a tag molecule to
form a tag molecule-analyte adduct having a density different from
the density of the analyte; c) providing a multi-phase system
comprising two or more phases with clear boundaries, wherein at
least one of the phases comprises a phase component, wherein the
phase component is selected from the group consisting of a polymer,
a surfactant, and combinations thereof; each of the two or more
phases has a different density and the two or more phases, taken
together, represent a density gradient; and the phases are
phase-separated from each other; and d) introducing the sample
comprising the tag molecule-analyte adduct to the multi-phase
system; and e) allowing the tag molecule-analyte adduct to migrate
to a location in the multiphase system that is characteristic of
its density, wherein during migration the sample contacts one or
more of the two or more phases sequentially and the analyte and the
tag molecule-analyte adduct occupy different locations. As used
herein, location means a position at, below, or above the interface
between phases.
[0019] In still another embodiment, the method comprises: a)
providing a sample comprising at least one analyte of interest; b)
providing a multi-phase system comprising two or more phases with
clear boundaries, wherein at least one of the phases comprises a
phase component, wherein the phase component is selected from the
group consisting of a polymer, a surfactant, and combinations
thereof; each of the two or more phases has a different density and
the two or more phases, taken together, represent a density
gradient; and the phases are phase-separated from each other; c)
introducing the sample comprising a tag molecule and the analyte to
the multi-phase system, wherein the tag molecule and the analyte
combine to form a tag molecule-analyte adduct having a density
different from the density of the analyte; and d) allowing the tag
molecule-analyte adduct to migrate to a location in the multiphase
system that is characteristic of its density, wherein during
migration the sample contacts one or more of the two or more phases
sequentially and the analyte and the tag molecule-analyte adduct
occupy different locations.
[0020] In another embodiment, a kit for separating a sample
comprising one or more biological analytes of interest using a
multi-phase system is disclosed. The kit comprises a) two or more
phase components selected from the group consisting of a polymer, a
surfactant, and combinations thereof; b) optionally a tag molecule
capable of binding the one or more biological analytes of interest,
wherein the tag molecule has a different density than the
biological analyte of interest; and c) instructions for: (i)
combining the two or more phase-separated solutions with a common
solvent to create a multi-phase system; (ii) optionally, combining
the biological analyte of interest and tag molecule, and (iii)
separating the biological analyte of interest from the sample.
[0021] In one or more embodiments, the kit further comprising an
aliquot of a common solvent which, when combined with the two or
more phase components, provides a multiphase system.
[0022] In one aspect, the kit comprises instructions which direct
that the biological analyte of interest be combined with the tag
molecule to form a tag molecule-analyte adduct before introduction
to the multi-phase system.
[0023] In another aspect, the kit comprises instructions that
further direct that the biological analyte of interest and tag
molecule be added to the multi-phase system to combine to form a
tag molecule-analyte adduct in the multi-phase system.
[0024] In another aspect, the kit further comprises one or more
additives selected from the group consisting of miscible
surfactants, salts, dyes, nutrients, vitamins, antibiotics,
anticoagulants, and buffers for combination with the phase
components.
[0025] The kit of claim 49, wherein the kit comprises a tag that
has an affinity for one or more analytes of interest.
[0026] In one or more aspects, the tag has an affinity for one or
more phase components.
[0027] In one or more aspects, the kit comprises a lytic agent for
introduction into one or more phases of the multiphase system.
[0028] In one or more of the proceeding embodiments, the at least
two phases share a common solvent.
[0029] In one or more of aspects, the multi-phase system is an
aqueous system and the common solvent is an aqueous solvent.
[0030] In one or more of the preceding embodiments, the multi-phase
system is a non-aqueous system and the common solvent is an organic
solvent.
[0031] In one or more embodiment, the phase components are selected
to be biologically compatible.
[0032] In one or more embodiments, the biological sample comprises
cells. In one aspect, the cells are selected from the group
consisting of animal, plant, protozoan, and prokaryotic cells.
[0033] In one or more embodiments, one or more phases comprise a
lysing agent to cause the cells to lyse, the biological analyte of
interest being recovered from cell lysate.
[0034] In one or more of the preceding embodiments, the biological
analyte is selected from the group consisting of organelles, cell
fragments, cell membranes, cell membrane fragments, viruses,
virus-like particles, bacteriophage, cytosolic proteins, secreted
proteins, signaling molecules, embedded proteins, nucleic
acid/protein complexes, organelles, minicells, nucleic acid
precipitants, chromosomes, nuclei, mitochondria, chloroplasts,
flagella, biominerals, protein complexes, protein aggregates, and
combinations thereof.
[0035] In one or more of the preceding embodiments, the biological
sample comprises one or more parasites selected from the group
consisting of worms, insects, protozoa, arachnids, and
arthropods.
[0036] In one or more of the preceding embodiments, the biological
sample comprises a biological fluid. In one or more aspects, the
biological sample is selected from the group consisting of food,
juice, and milk. In another aspect, the biological sample comprises
one or biological carriers selected from the group consisting of
whole blood, plasma, serum, sweat, feces, urine, saliva, tears,
vaginal fluid, prostatic fluid, gingival fluid, amniotic fluid,
intraocular fluid, cerebrospinal fluid, seminal fluid, sputum,
ascites fluid, pus, nasopharengal fluid, wound exudate fluid,
aqueous humour, vitreous humour, bile, cerumen, endolymph,
perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid,
sebum, vomit, and combinations thereof.
[0037] In one or more aspects, the biological sample is tested for
contaminants selected from the group consisting of pathogens,
pests, heavy metals, and pesticides.
[0038] In the embodiments of the preceding claims, biological
analyte is separated and analyzed to distinguish cell states
selected from the group consisting of normal cells, diseased cells,
parasitized cells, cancer cells, foreign cells, and infected
cells.
[0039] In one or more embodiments, the sample comprises a plurality
of analytes and each analyte migrates to a different location in
the phase-separated system.
[0040] In one or more embodiments, after migration, the analyte
resides at a boundary location.
[0041] In one or more of the preceding embodiments, the boundary
location is at an interface between a phase with a density greater
than the density of the analyte and a phase with a density that is
less than the density of the analyte.
[0042] In one or more aspects, after migration, the analyte resides
within a phase of the phase-separated system whose density matches
the density of the analyte.
[0043] In another aspect, the analyte/phase-separated system is
centrifuged to accelerate migration of the analyte.
[0044] In some aspects, the analyte migrates under gravitational
forces. In other aspects, the analyte migrates under buoyancy
forces.
[0045] In one or more of the preceding embodiments, the phase
separated system is supported in a column or test tube.
[0046] In one or more of the preceding embodiments, the phase
separated system is supported in a capillary tube, plastic test
tube, falcon tube, culture tube, well plates, cuvette, along a
filament, or on a sheet.
[0047] In one or more aspects, the sample comprises one or more
biological analytes of interest. In other aspects, the sample
comprises one or more non-biological analytes of interest.
[0048] In one or more aspects, the multi-phase system comprises one
or more biologically compatible phases.
[0049] In one or more aspects, the surfactant is selected from the
group consisting of polysorbate,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
("CHAPS"), polyoxyethylene-polyoxypropylene,
1-O-Octyl-.beta.-D-glucopyranoside,
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,
2-(Perfluoroalkyl)ethyl methacrylate, N,N-dimethyldodecylamine
N-oxide, polyethylene glycol dodecyl ether, sodium dodecyl sulfate,
sodium cholate, nonylphenol polyoxyethylene, benzylalkonium
chloride, and dodecyltrimethylammonium chloride.
[0050] In one or more aspects of the preceding embodiment, the
polymer is selected from the group consisting of dextran,
polysucrose, poly(vinyl alcohol), poly(2-ethyl-2-oxazoline),
poly(methacrylic acid), poly(ethylene glycol), polyacrylamide,
polyethyleneimine, hydroxyethyl cellulose, polyvinylpyrrolidone,
carboxy-polyacrylamide, poly(acrylic acid),
poly(2-acrylamido-2-methyl-1-propanesulfonic acid),
poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic
acid), polyallylamine, alginic acid, dextran sulfate, chondroitin
sulfate A, diethylaminoethyl-dextran,
poly(2-vinylpyridine-N-oxide), polydimethylsiloxane, and
polypropylene glycol). In some aspects, the polymer is selected
from the group of GRAS polymers. In one or more aspects, the
polymer is selected from the group of homopolymers, random
copolymers, block copolymers, graft copolymers, ter-polymers,
dendrimers, star polymers and combinations thereof. In still other
aspects, the polymer is linear, branched and/or cross-linked.
[0051] In one or more of the preceding embodiments, the method
comprises a system further comprising one or more additives
selected from the group consisting of miscible surfactants, salts,
dyes, nutrients, vitamins, antibiotics, anticoagulants, and
buffers.
[0052] In at least one aspect, the sample comprises the analyte of
interest and one or more impurities, the impurity having the same
density of the analyte, and the impurity having a different density
than the tag molecule-analyte adduct.
[0053] In one or more aspects, the analyte of interest has an
affinity for the tag molecule, and wherein the analyte of interest
and tag molecule preferentially link to form a tag molecule-analyte
adduct, the tag molecule-analyte adduct being linked by a method
selected from the group consisting of covalent bonding,
non-covalent bonding, hybridization, electrostatic interactions,
complexation, and conjugation.
[0054] In one or more aspect, the tag in the tag molecule-analyte
adduct has an affinity for one or more phase components, the tag of
the tag molecule-analyte adduct and the phase component
preferentially linking such that the tag molecule-analyte adduct
preferentially aggregates in one or more phases containing phase
components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The subject matter is described with reference to the
following figures, which are presented for the purpose of
illustration only and are not intended to be limiting of the
invention.
[0056] FIG. 1 shows separation of E. coli from the cellular
components of human whole blood using a PEG/Ficoll system with
density steps at approximately 1.030 and 1.101 g/ml in which a
strong increase in signal was seen when E. coli was added to blood
at a concentration of 10 6 CFU/mL.
[0057] FIG. 2 shows separation of E. coli from human whole blood
amplified by crystal violet A PEG/Ficoll system with density steps
at approximately 1.028 and 1.096 g/mL, which separated the denser
E. coli from the human blood components.
[0058] FIG. 3 A-C is an image showing the separation of whole blood
into erythrocytes, leukocytes, and cell debris at interfaces of the
MPS.
[0059] FIG. 4 shows images of a two-phase PEOZ/Ficoll system used
to separate cells and plasma from whole blood.
[0060] FIG. 5 shows an image of dyed density standards banding at
the polymer/polymer interface of the triphasic poly(vinyl
alcohol)/poly(ethylene glycol)/dextran system.
[0061] FIG. 6 shows the result of a separation of malaria-infected
red blood cells from healthy red blood cells.
[0062] FIG. 7 A-B shows separation of erythrocytes using a
two-phase AMPS as a malaria diagnostic.
[0063] FIG. 8 shows fluorescent images of CD4+ T cells (black) in
capillary tubes (.about.20 .mu.L total volume: 10 .mu.L polymer
system+10 .mu.L cell sample) labeled with a fluorescein-conjugated
antibody to CD4 in a two-phase system centrifuged at 16000 g for 2
minutes; cells that were more dense than both phases sedimented to
the bottom of the capillary tube (A); cells with a density between
the densities of the two adjacent phases were captured at the
interface between phases (B).
[0064] FIG. 9 shows a series of fluorescent images of two phase
systems with E. coli that has been genetically modified to express
red fluorescent protein (RFP); bacteria were captured at the
interface (tubes 1-3) or at the bottom (tubes 4-5).
[0065] FIG. 10 is a graph (n=7) of interface capture percentage as
a function of the density of a lower phase for erythrocytes, which
allows densities of cell types to be determined for designing MPS
to separate cell types selectively based on density.
[0066] FIG. 11 is a graph (n=3) showing the metabolic activity of
MD-MBA-231 breast cancer cells as a function of time and
concentration after separation using AMPS.
[0067] FIG. 12 is a graph showing the outcome of unique
two-component mixtures of twenty-three aqueous polymer solutions
and eleven aqueous surfactant solutions mixtures: no phase
separation (miscible; grey box), formation of a precipitate or a
gel (incompatible; black box), and phase separation (immiscible;
red box).
DETAILED DESCRIPTION
Introduction
[0068] The disclosed methods are used to separate objects or
impurities in samples according to the densities of the objects or
impurities, relative to the densities of the phases of a MPS.
Everything has a density. Thus, because the disclosed methods can
be used to separate, isolate, characterize, analyze, prepare, and
purify such diverse objects, the disclosed methods can be applied
to many contexts. For example, the disclosed methods can be used in
the forensics science context to separate and process objects of
interest from complex samples, e.g., to separate mixtures of
different types of biological samples (e.g., saliva or blood), or
to separate biological samples from non-biological samples (e.g.,
separating bone from rock and other debris). These methods can be
used to separate organisms from, or study organisms in seawater,
irrigation water, or mine effluent. For example, the disclosed
methods can be used with seawater to study small ocean organisms to
keep buoyant densities close to what they are in nature, or with
irrigation water or mine effluent to study the density effects on
micro-organisms when exposed to these liquids. The disclosed
methods can also be used monitor animal and plant health. Animal
tissues and plant material can be broken down to the cellular level
to detect cellular abnormalities indicative of disease and
infection. The disclosed methods of separating and analyzing
objects can also be used in pharmaceutical processing to detect and
quantify impurities. Similarly, these methods can be used to detect
contaminants such as pathogens, pests, heavy metals, and pesticides
in food processing to ensure quality control.
Multi-Phase System
[0069] MPS for use in the separation of biological analytes are
described. The multi-phase system comprises two or more phases
which are phase-separated from each other, wherein each of the
phases comprises a phase component. Each of the two or more phases
has a different density and the phases, taken together, represent a
density gradient, with the density of the phases increasing from
the top phase to the bottom phase of the MPS as the MPS is viewed
vertically. The phase component is a polymer, surfactant, or
combinations thereof. The phases in the MPS can be aqueous or
organic. In some embodiments, at least one phase of the MPS is
aqueous and at least one phase of the MPS is organic. In some
instances, such as when the sample contains biological analytes
that must be kept active or living, it may be desirable for the MPS
to comprise biologically compatible or substantially biologically
compatible phases and phase components. For example, the solution
can be an appropriately buffered aqueous solution and the phase
components are selected for biocompatibility.
[0070] In some embodiments, the multi-phase polymer system
comprises at least three phases. In some embodiments, the
multi-phase system comprises at least four phases. In some
embodiments, the multi-phase polymer system comprises at least five
phases. In some embodiments, the multi-phase polymer system
comprises at least six phases. Multi-phase systems with more phases
are contemplated. The MPS includes at least two phases with a
common solvent. However, when more than two phases are used, it is
possible to include phases using different solvents. It is also
possible to include phases that do not include a phase component,
such as aqueous or organic solvents, liquid polymers, liquid
metals, fluorinated liquids, and ionic liquids. Such variety
improves the ability of the system to separate complex samples.
[0071] The MPS used in the disclosed methods can comprise aqueous
phases, non-aqueous phases, or a combination of aqueous and
non-aqueous phases. In some embodiments, each of the adjacent
phases shares a common organic solvent. In some embodiments, each
of the two or more phases is organic. In some embodiments, each of
the two or more phases is aqueous. In some embodiments, the
multi-phase system comprises at least one aqueous phase and at
least one organic phase.
[0072] In some embodiments, the multi-phase system is aqueous and
each phase of the MPS comprises a phase component soluble in the
aqueous solvent. Non-limiting examples of aqueous solvent include
water, D.sub.2O, buffered water, e.g., phosphate buffers, cell
lysis buffer, cell culture medium, e.g., nutrient media, selective
media, transport media, enriched media, seawater, mine effluent,
and irrigation water. In some embodiments, the aqueous MPS can
comprise additional one or more organic phases comprising organic
solvents. The organic phase exhibits limited interaction with, and
phase-separated from, the aqueous polymer phases, however the phase
component including the organic phase is soluble in an organic
solvent.
[0073] In some specific embodiments, the multi-phase system further
comprises one or more additional phases selected from the group
consisting of organic solvents, ionic liquids, silicone oils,
organic oils, fluorinated liquids, and metals that are liquid at
room temperature. Suitable organic liquids include those that
exhibit limited interaction with water and will phase-separate from
the aqueous phases. Such additional phases are not required to have
a phase component.
[0074] In some embodiments, the multi-phase system is organic and
each phase of the MPS comprises a phase component dissolved in an
organic solvent. Non-limiting examples of organic solvent include
chloroform, ether, ethyl acetate, perfluorinated solvents, oils,
dichloromethane, tetrahydrofuran, toluene, tetrabromoethane,
methanol, dimethylsulfoxide, ethanol, supercritical CO.sub.2, fuel,
methanol, and lubricant. In some specific embodiments, the
different phases of the MPS comprise the same organic solvent. In
other specific embodiments, the different phases of the MPS
comprise different organic solvent. In other embodiments, the MPS
comprises a liquid polymer as one of the phases. Non-limiting
examples of liquid polymers include polydimethylsiloxane,
poly(propylene glycol), poly(ethyl vinyl ether), cis(polyisoprene),
polyethyleneimine, polybutadiene, polydimethylsiloxane,
poly(propylene glycol), poly(ethyl vinyl ether), cis(polyisoprene)
and polysorbate (herein referred to by the trade name "Tween").
[0075] In some embodiments, the MPS comprises at least an aqueous
phase and at least one organic phase. Each phase may comprise a
phase component and the mixture of aqueous and organic phases,
taken together, represent a density gradient.
[0076] One component of an MPS can be a polymer. Non-limiting
examples of polymers include GRAS polymers, dextran, polysucrose
(herein referred to by the trade name "Ficoll"), poly(vinyl
alcohol), poly(2-ethyl-2-oxazoline), poly(methacrylic acid),
poly(ethylene glycol), polyacrylamide, polyethyleneimine,
hydroxyethyl cellulose, polyvinylpyrrolidone,
carboxy-polyacrylamide, poly(acrylic acid),
poly(2-acrylamido-2-methyl-1-propanesulfonic acid),
poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic
acid), polyallylamine, alginic acid, poly(2-vinylpyridine-N-oxide),
diethylaminoethyl dextran, dextran sulfate, chondroitin sulfate A,
polydimethylsiloxane, and poly(propylene glycol). In one or more
aspects the polymer is selected from the group consisting of
wherein the surfactant is selected from the group consisting of
dextran, polysucrose, poly(vinyl alcohol),
poly(2-ethyl-2-oxazoline), poly(methacrylic acid), poly(ethylene
glycol), polyacrylamide, polyethyleneimine, hydroxyethyl cellulose,
polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylic acid),
poly(2-acrylamido-2-methyl-1-propanesulfonic acid),
poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic
acid), polyallylamine, alginic acid, dextran sulfate, chondroitin
sulfate A, diethylaminoethyl-dextran,
poly(2-vinylpyridine-N-oxide), polydimethylsiloxane, and
poly(propylene glycol). In some embodiments, the polymer is
selected from the group consisting of homopolymer, block copolymer,
random copolymer, copolymer, terpolymer, and combinations thereof.
In some embodiments, the polymer has a morphology selected from a
group consisting of linear polymer, branched polymer, co-polymer,
cross linked polymer, and dendrimer system. In some embodiments,
the MPS comprises a polymer soluble in the solvent. In other
embodiments, the MPS comprises a polymer soluble in water.
[0077] One component of an MPS can be a surfactant. Non-limiting
examples surfactants that can be used to modify surface tension
include Tween, CHAPS, polyoxyethylene-polyoxypropylene (herein
referred to by the trade name "Pluronic F68"),
1-O-Octyl-.beta.-D-glucopyranoside,
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (herein
referred to by the trade name "Triton"), 2-(Perfluoroalkyl)ethyl
methacrylate (herein referred to by the trade name "Zonyl"),
N,N-dimethyldodecylamine N-oxide, polyethylene glycol dodecyl ether
(herein referred to by the trade name "Brij"), sodium dodecyl
sulfate, nonylphenol polyoxyethylene, sodium cholate,
benzylalkonium chloride, and dodecyltrimethylammonium chloride. In
some specific embodiments, surfactant phases comprising Pluronic
F68 and CHAPS are selected to form an aqueous multi-phase polymer
system with one or more aqueous polymer phases. Non-limiting
examples of the polymer used in these embodiments include
poly(methacrylic acid), poly(2-ethyl-2-oxazoline), dextran, Ficoll,
polyacrylamide, and polyethyleneimine. In one or more aspects, the
surfactant is selected from the group consisting of Tween 20,
CHAPS, Pluronic F68, 1-O-Octyl-.beta.-D-glucopyranoside, Triton,
Zonyl, N,N-dimethyldodecylamine N-oxide, Brij, sodium dodecyl
sulfate, sodium cholate, nonylphenol polyoxyethylene,
benzylalkonium chloride, and dodecyltrimethylammonium chloride. The
use of the surfactant can provide additional aqueous phases and
facilitate the formation of the MPS. Other appropriate surfactants
to accomplish this objective can be selected by the persons of
ordinary skills in the art.
[0078] The aqueous surfactant phase and the aqueous polymer phase
exhibit limited interaction and thus phase-separate. In some
embodiments, more than one additional aqueous surfactant phase can
be added to the aqueous multi-phase polymer system. In some
specific embodiments, two aqueous surfactant phases and one or more
aqueous polymer phases phase-separate and form an aqueous
multi-phase polymer system.
[0079] In some aspects, one or more of the phases of the MPS
comprises light or heavy salts one or more salts that aid in the
phase-separation process. The salts dissolve in the phase,
resulting in a change of the phase density, ionic strength or
solubility of the phase component in the phase solvent.
Non-limiting examples of salt include sodium chloride, potassium
chloride, sulfates, phosphates, nitrites, citrates, EDTA, heparin,
acids (e.g., HCl), bases (e.g., NaOH), glycine, buffer salts (e.g.,
tris(hydroxymethyl)aminomethane), acetates, and sulfonates. In some
embodiments, salt(s) can be added to the polymer systems in order
to adjust the density, pH, and/or osmolality of the multiphase
systems.
[0080] In some embodiments, small molecules can be added for some
specific functions. For example, in some specific embodiments,
heparin or sodium EDTA are added as an anticoagulant. In some other
embodiments, sodium benzoate is added as a preservative.
[0081] In one or more embodiments, particularly multi-phase systems
designed for use with more than two phase components, one or more
polymers or surfactants that do not phase separate with each of the
other phase components can be used as additives to modify the
density, viscosity, osmolality, or refractive index of the phase
component in which the additive resides. The polymers or
surfactants are added to the various phases of the multi-phase
system in addition to the phase components at concentrations less
than is required to phase separate into a separate phase. In this
instance, the surfactant performs the functions that are typical of
surfactants, such as modify the surface tension of the
solution.
[0082] Non-limiting examples of other additives that can be
included in the phases include used in formulations to produce
aggregation include, organic additives such as dyes and reactive or
non-reactive dissolved gasses and cosolvents. In addition, the
additives can be colloids or micelles. The phase components are
selected so that the resulting phases exhibit limited interaction
and thus are phase-separated from each other. Phase-separation
refers to the phenomena where two or more phases, each comprising a
phase component, when mixed together, form the same number of
distinct phases where each phase has clear boundaries and is
separated from other phases. Each phase component used in the
solution is selected to be soluble in the solvent of the phase, so
that each phase is phase-separated from other adjacent phase(s).
When the multi-phase polymer system is designed, each phase
component is selected to predominantly reside in one particular
phase of the multi-phase system. It should be noted that in the
resulting multi-phase system, every phase could contain varying
amounts of other phase components from other phases in the MPS, in
addition to the selected desired phase component in that phase.
Unless otherwise specified, the phase component composition in each
phase of the multi-phase system recited herein generally refers to
the starting phase component composition of each phase, or to the
predominant phase component composition of each phase. In some
embodiments, the phase component composition on a phase component
comprises predominantly one phase component and small amount of one
or more other phase components. In some embodiments, the phase
component composition in a phase comprises about 70% of one phase
component. In some embodiments, the phase component composition in
a polymer phase comprises about 75% of one phase component. In some
embodiments, the phase component composition in a phase comprises
about 80% of one phase component. In some embodiments, the phase
component composition on a phase comprises about 85% of one phase
component by weight. In some embodiments, the phase component
composition on a phase comprises about 90% of one phase component
by weight. In some embodiments, the phase component composition in
a phase comprises about 95% of one phase component. In some
embodiments, the phase component composition in a phase comprises
about 99% of one phase component by weight. Other combinations of
phase component compositions are contemplated.
[0083] In some embodiments, the concentration of the phase
component in the phase is from about 0.1% to about 50% (wt/vol). In
some embodiments, the concentration of the phase component in the
phase is from about 0.5% to about 40% (wt/vol). In some
embodiments, the concentration of the phase component in the phase
is from about 1% to about 20% (wt/vol). In some embodiments, the
concentration of the phase component in the phase is from about 5%
to about 10% (wt/vol). In some embodiments, the concentration of
the phase component in the phase is about 10% (wt/vol). In some
embodiments, the concentration of the phase component in the phase
is about 15% (wt/vol). In some embodiments, the composition or
density of the resulting phases in the multi-phase system could be
affected by the starting concentration of the phase component
phases.
[0084] In some embodiments, one or more of the phases of the MPS
are degassed to remove residual amount of gas dissolved in the
phases. In some embodiments, the phases are degassed to remove
oxygen from the phase to avoid possible oxidation of the sample
applied onto the MPS.
[0085] Various types of form factors of the MPS can be used. In
some embodiments, the MPS is contained in a tube, column, vial,
well plate, container, bottle, drum, porous film, capillary tube,
Eppendorf tube (plastic test tube), falcon tube, culture tube, well
plates, cuvette or sponge. In other embodiments, the MPS is
deposited on paper. In still other embodiments, the MPS is
deposited on cloth or string.
[0086] Generally speaking, if a combination of multiple phase
component phases results in a phase-separated MPS, any
sub-combination of the multiple phase component phases will also
result in a phase-separated MPS. Thus, if a five-phase component
MPS phase-separates, any four-polymer aqueous system selected from
the five phase component phases can also phase-separate. Likewise,
any two- or three-phase component MPS selected from the five phase
component phases can also phase-separate. Other suitable
combinations of polymers are contemplated.
[0087] In some embodiments, whether or not a MPS comprising
multiple phase components will phase-separate can be predicted
based on the properties of the MPS containing the sub-combination
of the multiple phase components. For instance, phase solutions
containing phase components A, B, and C, respectively, will
phase-separate and form a three-phase MPS if the phase component A
solution and phase component B solution phase-separate, the phase
component A solution and phase component C solution phase-separate,
the phase component B solution and phase component C solution
phase-separate. Similarly, solutions of phase components A, B, C,
and D will form a four-phase MPS if the following phase components
combinations all phase-separate: A-B-C, A-B-D, A-C-D, and B-C-D.
Likewise, solutions of phase components A, B, C, D, and E will form
a five-phase MPS if the following phase components combinations all
phase-separate: A-B-C-D, A-B-C-E, A-B-D-E, A-C-D-E, B-C-D-E. Also,
solutions of phase components A, B, C, D, E, and F will form a
six-phase MPS if the following phase components combinations all
phase-separate: A-B-C-D-E, A-B-C-D-F, A-B-C-E-F, A-B-D-E-F,
A-C-D-E-F, and B-C-D-E-F. The predictions of more complex MPS based
on the same principle is contemplated. These predictions have
largely been confirmed by experimental data. Certain predicted MPS
have not been produced by experiments can be produced via routine
experimental optimization.
[0088] As used herein, a MPS can be identified by its phase
components in the phases of the MPS. For instance, a
Ficoll-dextran-poly(2-ethyl-2-oxazoline) system refers to a
three-phase MPS, wherein the phase components in each phases of the
MPS are Ficoll, dextran, and poly(2-ethyl-2-oxazoline),
respectively. Each phase includes a suitable solvent capable of
dissolving the phase components. In some instances, a liquid
polymer is used and the liquid polymer forms a phase with no
solvent added.
[0089] In some embodiments, the multi-phase polymer system is
provided by mixing suitable polymers or surfactants with a solvent
and subjecting the mixture to centrifugation. Any types of
centrifugation known in the art can be used in the formation of the
MPS. In some embodiments, the MPS is formed using soft
centrifugation. Soft centrifugation is described above. In some
specific embodiments, the soft centrifugation is achieved by an
eggbeater centrifuge. Other methods of soft centrifugation known in
the art are also contemplated.
Density-Based Separation Using Multi-Phase Systems
[0090] A method of using a MPS comprising two or more phases to
separate samples comprising analytes of interest according to the
densities is described. In some embodiments, a method of analyzing
or separating a sample comprising one or more analytes of interest
according to the density of the analytes or the density of other
elements comprising the sample using a MPS is used. The method
comprises: a) providing a multi-phase system comprising two or more
phase-separated solutions, wherein each phase comprises a phase
component selected from the group consisting of a polymer, a
surfactant and combinations thereof, wherein at least one phase
comprises a polymer; each said phase has an upper and a lower phase
boundary; and each of the two or more phases has a different
density and the phases, taken together, represent a density
gradient; and b) introducing a biological sample comprising one or
more biological analytes of interest without disrupting the
phase-separated solution; and c) allowing each of the biological
analytes to migrate to a location in the multi-phase system that is
characteristic of its density, wherein during migration the sample
contacts one or more of the two or more phases sequentially. The
analytes may have different densities. Such differences in density
can result in the analyte preferentially accumulating in one of the
phases in the MPS or more typically, at a phase interface or
boundary. The desired analyte in the sample can then be viewed,
wherein information regarding the analyte is obtained based on its
location within the multi-phase system. In other embodiments, the
analyte is recovered, thus resulting in an improved purity or
isolation of such analyte.
[0091] In other embodiments, a method of analyzing a sample
comprising at least one analyte of interest according to the
density of a tagged analyte of interest in an MPS is disclosed.
[0092] The method comprises: a) providing a sample comprising at
least one analyte of interest; b) combining the sample with a tag
molecule to form a tag molecule-analyte adduct having a density
different from the density of the analyte; c) providing a
multi-phase system comprising two or more phases with clear
boundaries, wherein at least one of the phases comprises a phase
component, wherein the phase component is selected from the group
consisting of a polymer, a surfactant, and combinations thereof;
each of the two or more phases has a different density and the two
or more phases, taken together, represent a density gradient; and
the phases are phase-separated from each other; and d) introducing
the sample comprising the tag molecule-analyte adduct to the
multi-phase system; and e) allowing the tag molecule-analyte adduct
to migrate to a location in the multiphase system that is
characteristic of its density, wherein during migration the sample
contacts one or more of the two or more phases sequentially and the
analyte and the tag molecule-analyte adduct occupy different
locations. As used herein, location means a position at, below, or
above the interface between phases.
[0093] In another embodiment, the method comprises: a) providing a
sample comprising at least one analyte of interest; b) providing a
multi-phase system comprising two or more phases with clear
boundaries, wherein at least one of the phases comprises a phase
component,
[0094] wherein the phase component is selected from the group
consisting of a polymer, a surfactant, and combinations thereof;
each of the two or more phases has a different density and the two
or more phases, taken together, represent a density gradient; and
the phases are phase-separated from each other; c) introducing the
sample comprising a tag molecule and the analyte to the multi-phase
system, wherein the tag molecule and the analyte combine to form a
tag molecule-analyte adduct having a density different from the
density of the analyte; and d) allowing the tag molecule-analyte
adduct to migrate to a location in the multiphase system that is
characteristic of its density, wherein during migration the sample
contacts one or more of the two or more phases sequentially and the
analyte and the tag molecule-analyte adduct occupy different
locations.
[0095] In another embodiment, a kit for separating a sample
comprising one or more biological analytes of interest using a
multi-phase system is disclosed. The kit comprises a) two or more
phase components selected from the group consisting of a polymer, a
surfactant, and combinations thereof; b) optionally a tag molecule
capable of binding the one or more biological analytes of interest,
wherein the tag molecule has a different density than the
biological analyte of interest; and c) instructions for: (i)
combining the two or more phase-separated solutions with a common
solvent to create a multi-phase system; (ii) optionally, combining
the biological analyte of interest and tag molecule, and (iii)
separating the biological analyte of interest from the sample.
[0096] In one or more embodiments, the kit further comprising an
aliquot of a common solvent which, when combined with the two or
more phase components, provides a multiphase system.
[0097] In one aspect, the kit comprises instructions which direct
that the biological analyte of interest be combined with the tag
molecule to form a tag molecule-analyte adduct before introduction
to the multi-phase system.
[0098] In each of these embodiments, the analyte (or analyte-tag
adduct) traverses the phase(s) of the MPS sequentially and migrate
to a location of MPS corresponding to its density. In this process,
the analyte does not have simultaneous contact with two or more
phases of the MPS except when passing the interface between two
adjacent phases. This method is distinguished from the separation
based on affinity as described above in that in the latter, the
analyte needs to have simultaneous contact with all of the phases
of the MPS so that a thermodynamic equilibrium is reached and the
analyte can preferentially reside in one of the phases based on its
affinity towards that phase. This process is commonly referred to
as `partitioning` or `extraction.` In comparison, in the
density-based separation as described herein, the analyte migrates
through the MPS phase one at a time, contacting one or more of the
phases sequentially and eventually arriving at a location in the
MPS characteristic of its density. Because the analyte only
contacts a single phase at a time, except at the interface where
the analyte may contact and interact with two adjacent phases
simultaneously, no partitioning or extraction of the analyte into a
particular phase is possible.
[0099] Each phase of the MPS has an upper and a lower phase
boundary, and two adjacent phases forms a common interface in
between. In most instances, there is not an exact match between the
analyte density and the density of any particular phase. The
analyte's density is between the densities of two adjacent phases
in a MPS, and the analyte should therefore remain at the interface
of the two adjacent phases. If the analyte should have the same
density as that of one of the phases, the analyte will remain with
in the density-matched phase without contacting any boundary. In
this case, the analyte resides within the phase due to a density
match and not due to any favorable or preferential interaction of
the analyte with one phase over another. In still other
embodiments, the analyte may have a density less than that of the
top phase of the MPS (the phase with the least density) and remain
at the top of the MPS with a portion of the analyte above the upper
boundary of the top phase after migration. In still other
embodiments, the analyte may have a greater density than that of
the bottom phase of the MPS (the phase with the most density) and
remain at the bottom of the MPS after migration.
[0100] In some embodiments, the analyte is allowed to migrate based
on gravity. In other embodiments, the analyte is allowed to migrate
using a centrifuge. Non-limiting examples of centrifuge include
laboratory centrifuges (using either a fixed angle or swinging
bucket rotors) and soft-centrifuges. Soft centrifugation refers the
uses of soft tubing, e.g., polyethylene tubing, as the sample
container and a simple device as the rotor (see, Wong et al., "Egg
beater as centrifuge: isolating human blood plasma from whole blood
in resource-poor setting", Lab Chip, 2008, 8, 2032-2037). In some
specific embodiments, the soft centrifugation is achieved by an
eggbeater centrifuge. Other methods of soft centrifugation known in
the art are also contemplated.
[0101] Migration occurs until the analyte reaches a phase with
which it is density matched or a phase of higher density, so that
it no longer moves over time through the multi-phase system. This
situation is referred to as having reached an `equilibrium
location`. In the case of gravity migration, the time to reach
equilibrium migration is in the range of seconds to several days
depending on factors such as viscosity, particle size, and buoyant
density. Use of centrifugation can accelerate the migration process
and reduce the time to reach equilibrium location to minutes or
hours. The centrifuge works using the sedimentation principle,
where the centripetal acceleration causes more dense substances to
separate out along the radial direction (towards the bottom of the
tube). By the same token, less dense objects will tend to move to
the top. Centrifugation conditions can include speeds ranging from
1 g to over 170,000 g for periods lasting a few seconds to several
days. Centrifuges can be used with heating and cooling to, e.g.,
modify the viscosity of the system or to maintain cell viability.
Persons of ordinary skill in the art would know how to adjust these
conditions to achieve separation in a given system. For example,
speeds and times can be varied depending on the type of analyte
sought to be separated (pelleting application), complexity of the
sample, or composition of the MPS.
[0102] In some embodiments, a two-phase MPS can be used for
purposes disclosed herein. In some other embodiments, three or more
phase systems are used. It was believed that the inclusion of
additional phases may prevent the enrichment of the target molecule
in a specific phase, because the target molecule may distribute
into the additional phases. This belief may account for the lack of
literature regarding these multi-phase polymer systems. Applicants
have surprisingly found that broadening the landscape of polymers
that exhibit limited interaction in aqueous multi-phase polymer
systems provides superior tunability for applications based on
differences in density and affinity and finer control over the
partitioning of complex mixtures of objects.
[0103] The analytes used in the disclosed methods can be biological
in origin. The methods disclosed herein can be used for separating
biomoleculaes e.g., proteins, saccharides, polyterpenes,
polynucleic acids, extracting recombinant proteins, analyzing
enzymatic digestions, and partitioning cells. Other uses known in
the art are contemplated. Types of biological analytes that can be
used include, without limitation, cells, cancer cells, stem cells,
cell extracts, tissue extracts, cell organelles, cell fragments,
cell membranes, cell membrane fragments, viruses, virus-like
particles, bacteriophage, cytosolic proteins, secreted proteins,
signaling molecules, embedded proteins, nucleic acid/protein
complexes, nucleic acid precipitants, chromosomes, nuclei,
mitochondria, chloroplasts, flagella, biominerals, protein
complexes, phage, minicells, and protein aggregates, tissues,
organisms, small molecules, large-sized molecules, e.g.,
biomolecules including proteins, and particles. In one or more
aspects, the types of cells used in the disclosed methods include
mammalian cells selected from the group consisting of gland cells
(e.g., exocrine secretory epithelial cells, salivary gland mucous
cells, salivary gland serous cells, Von Ebner's gland cells,
mammary gland cells, lacrimal gland cells, ceruminous gland cells,
eccrine sweat gland dark cells, eccrine sweat gland clear cells,
apocrine sweat gland cells, gland of Moll cells, aebaceous gland
cells, Bowman's gland cells, Brunner's gland cells, seminal vesicle
cells, prostate gland cells, bulbourethral gland cells, bartholin's
gland cells, gland of littre cells, uterine endometrial cells,
isolated goblet cells, stomach lining mucous cells, gastric gland
zymogenic cells, gastric gland oxyntic cells, pancreatic acinar
cells, paneth cells, type II pneumocyte cells, and Clara cells),
hormone secreting cells (e.g., anterior pituitary cells,
intermediate pituitary cells, magnocellular neurosecretory cells,
gut and respiratory tract cells, thyroid gland cells, parathyroid
gland cells, adrenal gland cells, chromaffin cells, Leydig theca
interna cells, corpus luteum cells, granulosa lutein cells, theca
lutein cells, juxtaglomerular cells, racula densa cells, peripolar
cells, and mesangial cells), epithelial cells lining closed
internal body cavities (e.g., blood vessel and lymphatic vascular
endothelial fenestrated cells, blood vessel and lymphatic vascular
endothelial continuous cells, blood vessel and lymphatic vascular
endothelial splenic cells, synovial cells, serosal cells, squamous
cells, columnar cells, dark cells, vestibular membrane cells, stria
vascularis basal cells, stria vascularis marginal cells, Claudius
cells, Boettcher cells, choroid plexus cells, pia-arachnoid
squamous cells, pigmented and non-pigmented ciliary epithelial
cells, corneal endothelial cells, and peg cells), ciliated cells of
the respiratory tract cells, oviduct cells, uterine endometrium
cells, rete testis cells, and ductulus efferens cells, ciliated
ependymal cells of central nervous system, keratinizing epithelial
cells (e.g., epidermal keratinocyte, epidermal basal cells,
keratinocytes, nail bed basal cells, medullary hair shaft cells,
cortical hair shaft cells, cuticular hair shaft cells, cuticular
hair root sheath cells, hair root sheath cell of Huxley's layer,
hair root sheath cell of Henle's layer, external hair root sheath
cells, and hair matrix cells), wet stratified barrier epithelial
cells (e.g., surface epithelial cell of stratified squamous
epithelium of cornea, tongue, oral cavity, esophagus, anal canal,
distal urethra and vagina; basal cell of epithelia of the cornea,
tongue, oral cavity, esophagus, anal canal, distal urethra, and
vagina; and urinary epithelium cells), cells of the nervous system
(e.g., sensory transducer cells, auditory inner hair cell of organ
of corti, auditory outer hair cell of organ of corti, basal cell of
olfactory epithelium, cold-sensitive primary sensory neurons,
heat-sensitive primary sensory neurons, Merkel cell of epidermis,
olfactory receptor neurons, pain-sensitive primary sensory neurons,
photoreceptor cells of the retina, proprioceptive primary sensory
neurons, touch-sensitive primary sensory neurons, cholinergic
neurons, adrenergic neurons, peptidergic neural cells, inner and
outer pillar cells, inner and outer phalangeal cells, border cells,
hensen cells, taste bud supporting cells, olfactory epithelium
supporting cells, Schwann cells, satellite cells, enteric glial
cells, central nervous system neural and glial cells, and lens
cells), hepatocyte, adipocytes, liver lipocytes, kidney cells
(e.g., glomerulus parietal cells, glomerulus podocyte cells,
proximal tubule brush border loop of Henle thin segment cells,
distal tubule cells, and collecting duct cells), lung cells, Type I
pneumocytes, pancreatic duct cells, nonstriated duct cells,
principal cells, intercalated cells, duct cells, intestinal brush
border cells, exocrine gland striated duct cells, gall bladder
epithelial cells, ductulus efferens nonciliated cells, epididymal
principal cells, epididymal basal cells, extracellular matrix
cells, ameloblast epithelial cells, planum semilunatum epithelial
cells, loose connective tissue fibroblasts, corneal fibroblasts,
tendon fibroblasts, bone marrow reticular tissue fibroblasts,
nucleus pulpous cells, cementoblast/cementocytes,
odontoblast/odontocytes, hyaline cartilage chondrocytes,
fibrocartilage chondrocytes, fibroblast cartilage chondrocytes,
osteoblast/osteocytes, osteoprogenitor cells, hyalocytes of
vitreous body of eye, stellate cells of perilymphatic space of ear,
hepatic stellate cells, pancreatic stele cells, contractile cells,
skeletal muscle cells, heart muscle cells, smooth muscle cells,
blood and immune cells (e.g., erythrocyte, megakaryocyte, monocyte,
connective tissue macrophage, epidermal langerhans, osteoclast,
dendritic cell, microglial cell, neutrophil granulocyte, eosinophil
granulocyte, basophil granulocyte, mast cell, T cell, suppressor T
cell, cytotoxic T cell, natural killer T cell, B cell, and
reticulocyte), Stem cells and committed progenitors for the blood
and immune system (e.g., pigment cells, melanocytes, and retinal
pigmented epithelial cells), germ cells (e.g., oocyte, spermatid,
spermatocyte, spermatogonium cell, and spermatozoon, nurse cells
(e.g., ovarian follicle cell, and sertoli cells, and thymus
epithelial cells), interstitial cells, and combinations
thereof.
[0104] In one or more aspects, the type of cell used in the
disclosed methods is a plant cell selected from the group
consisting of parenchyma cells, chlorenchyma cells, collenchyma
cells, sclerenchyma, epidermal cells, cork cells, xylem cells,
xylem vessel cells, meristematic cells, and combinations
thereof.
[0105] In one or more aspects, the type of cell used in the
disclosed methods is a protozoan selected from the group consisting
of Amoeba, Paramecium, Euglena, and combinations thereof.
[0106] In one or more aspects, cells used in the disclosed methods
are prokaryotic cells selected from the group consisting of
bacteria (e.g., bacteria of the phyla Aquificae, Xenobacteria,
Fibrobacter, Bacteroids, Firmicutes, Planctomycetes, Chrysogenetic,
Cyanobacteria, Thermomicrobia, Chlorobia, Proteobacteria,
Spirochaetes, Flavobacteria, Fusobacteria, and Verrucomicrobia),
archaea (e.g., Crenarchaeota, Euryarchaeota, Korarchaeota,
Nanoarchaeota, Thaumarchaeota, Aigarchaeota), and combinations
thereof.
[0107] In one or more aspects, the analyte of interest is a virus
or virus-like particle selected from the group consisting of dsDNA
viruses, ssDNA viruses, dsRNA viruses, (+) ssRNA viruses, (-) ssRNA
viruses, ssRNA-RT viruses, dsDNA-RT viruses, and combinations
thereof.
[0108] In one or more aspects, tissues used in the disclosed
methods are mammalian tissues selected from the group consisting of
connective tissue (e.g., bone, cartilage, lymphoid tissue, blood,
areolar tissue, adipose tissue, elastic tissue, hypodermis, lamina
propria, submucosa, mesentery, fascia, muscle capsule, tendons,
sclera, and dermis), muscle tissue (e.g., smooth muscle, cardiac
muscle, and skeletal muscle), nervous tissue, epithelial tissue
(e.g., stratified squamous epithelium, cuboidal epithelium,
endothelium, simple columnar epithelium, simple squamous
epithelium, mesothelium, pseudostratified epithelium, transitional
epithelium, and glandular epithelium), tissues of the eye, ear, and
skin, and combinations thereof.
[0109] In one or more aspects, the types of tissue extracts used in
the disclosed method are extracts from tissues including occular,
gingival, heart, liver, brain, stomach, kidney, lung, gall bladder,
spleen, intestinal, uterine, prostatic, epithelial, connective, and
muscle tissues.
[0110] In one or more aspects, tissues used in the disclosed
methods are plant tissues selected from the group consisting of
vascular tissue, dermal tissue, ground tissue, meristematic tissue,
and combinations thereof.
[0111] In one or more aspects, the types of organisms used in the
disclosed method include parasites. In one or more aspects, the
parasite is a worm. Non-limiting examples of worms that can be
separated from biological samples include flat worms such as flukes
and tape worms, or roundworms such as eye worms, filarial worms,
Trichinella roundworms, hookworms, annelids ("ringed worms") such
as leeches, and ascarids. In another aspect, the disclosed methods
can be used to separate parasitic insects from samples.
Non-limiting examples of such insects include mosquitoes, fleas,
lice, ticks and mites.
[0112] In one or more aspects, the disclosed method is used to
analyze biological carriers, including without limitation, food,
juice, milk, whole blood, plasma, serum, sweat, feces, urine,
saliva, tears, vaginal fluid, prostatic fluid, gingival fluid,
amniotic fluid, intraocular fluid, cerebrospinal fluid, seminal
fluid, sputum, ascites fluid, pus, nasopharengal fluid, wound
exudate fluid, aqueous humour, vitreous humour, bile, cerumen,
endolymph, perilymph, gastric juice, mucus, peritoneal fluid,
pleural fluid, sebum, vomit, and combinations thereof. In such
biological carriers, it is possible to identify the presence or
absence of cells, cellular components, large biomolecules or other
components that can be an indication of disease, genetic condition
or infection.
[0113] Samples can be introduced to the MPS in the form of a
solution or suspension of material. Non-limiting examples of ways
in which these samples can be added to the MPS include by pour,
pipette, injection, drip, siphon, capillary action, spray,
aspiration followed by expulsion, and pump.
[0114] Because the separation is carried out using gravity
(enhanced gravitational force using centrifugation), the analyte is
desirably in suspension in the MPS phases, e.g., the analyte is
insoluble in the MPS phases. In addition, separation will be
achieved more readily and in a shorter time frame for larger
analytes. Without additional modifications, such a density tagging,
biomolecules of a size such that their interactions in solution are
predominantly gravity driven can be distinguished in the MPS
system. When analytes are sufficiently small, the molecular forces
(electrostatic, Van der Waals, etc) are sufficient strong relative
to the size and mass of the analyte that the interaction with the
solvent is dictated by these forces. Such analytes are capable
therefore of partitioning selectively into one or another phase due
to favorable molecular interactions that effectively disregard the
densities of the analytes. In most instances, the analyte has at
least one dimension that is greater than 200 nm, or greater than
500 nm, or greater than 1 .mu.m, which is sufficient for
gravitation forces to predominate.
[0115] In embodiments in which the sample comprises small particles
that are of interest, the samples can be subjected to aggregating
agents to induce aggregation of the small particles, so that the
analyte is larger and can be separated readily using MPS, or the
densities of the small particles can be modified using additives to
force their migration to different locations in the MPS such that
the aggregated small particles pass through one or more phases
sequentially. Non-limiting examples of small particles that require
aggregation include multivalent particles (e.g., viruses, cells,
and any basic unit that expresses surface markers), platelets
(adenosine diphosphate), and erythrocytes (hemagglutinin or
concanavalin A), As a result of the disclosed methods, one or more
analytes of interest may preferentially accumulate in one of the
phase or at an interface in the MPS, while another analyte,
impurity or object in the sample containing the analyte may
preferentially accumulate in another phase or interface of the MPS.
The desired analyte in the sample can be visualized after
separation via a variety of methods. Firstly, separation of some
analytes can visualized by human eye. Those that are not readily
visible by the eye can be visualized using methods known in the
art. For example, separation can be visualized with the aid of a
microscope, scanner, magnifying glass, fluorescence (e.g.,
fluorescein, rhodamine, and diamidino-2-phenylindole) and dye (e.g.
crystal violet, methylene blue, and acid orange). In addition, for
embodiments in which the analyte is a cell, a cell suspension can
be incubated in a solution of antibodies labeled with fluorophores
specific for cell type separated using the disclosed methods. In
another aspect in which the analyte is a bacterium, a suspension of
bacteria can be incubated with bacteriophage (a species-specific
virus) that are genetically modified to produce a fluorescent
protein (e.g., red fluorescent protein) within the bacterium.
[0116] In some embodiments, it will be sufficient to simply observe
the location of the analyte in the multi-phase system in order to
obtain useful information. For example, a biological sample such as
blood, urine or feces can be subjected to analysis by MPS in order
to determine the presence or absence of certain markers that can be
an indication of disease, genetic disorder or infection. For
example, the cellular components of blood can be separated by MPS
(Blood plasma has a density of about 1.025 g/cm.sup.3 and red blood
cells have a density of approximately 1.100 g/cm.sup.3. There is
some debate regarding the actual density of erythrocytes, however
they have been described with a narrow distribution of densities,
which makes detection of shifts in density useful for assessing
blood cell health.). The presence of blood disorders, such as
sickle cell anemia, can be inferred by the presence of an analyte
at a boundary that is specific to the density of sickle cell
erythrocytes as compared to healthy erythrocytes. Similarly, a
urinary infection could be detected by noting the presence of
analyte at a boundary that is selective for white blood cells.
Differences in densities that can be detected can be 0.001
g/cm.sup.3 or more, which is sufficient to detect such density
differences. Non-limiting examples of tests that require only
information about the location of the analyte in the MPS include
white blood cells in urine, sickle cell erythrocytes, parasitized
cells, parasitic organisms, and the presence or absence of rare
cells, diseased cells, foreign cells, bacteria, yeast, fungi, or
viruses.
[0117] MPS are selected to have phases with densities that are
capable of distinguishing between the analyte of interest and other
components of the sample. For the separation of blood plasma and
red blood cells, for example, phases having densities that are (1)
less than blood plasma, (2) greater than blood plasma, but less
than red blood cells and (3) greater than red blood cells could be
used to separate plasma cells and red blood cells into two
different boundary locations. A number of MPS systems with a range
of densities are known and can be used to practice the methods of
the invention. See, PCT Patent Application No. ______, filed on
Aug. 19, 2011, entitled "MULTIPHASE SYSTEMS AND USES THEREOF,"
identified by attorney docket number 0042697.251WO1.
[0118] In other embodiments, the desired analyte in the sample can
be recovered by retrieving the phase in which the analyte
preferentially has accumulated, thus resulting in an improved
purity of such analyte. Analytes can be recovered from the system
using extraction methods known in the art. In several aspects of
one or more embodiments, analytes retained in gradients can be
recovered using a fractionator, pipette, drip method,
side-puncturing a tube, or combinations thereof. In one aspect, a
fractionator can be used to carefully control the pressure on the
liquid and pull known volumes of the gradient in certain
increments. The drip method can also be used to extract separated
analytes. The bottom of a tube is punctured and allowed to drip
into sample tubes. This method, like the fractionator method, is
ideal for systems such as the disclosed MPS that form clear visual
interfaces that can be observed by eye. In another aspect, a
pipette is introduced to the top of the sample to remove most, but
not all, of the top layer without pulling too close to the
interface. Once the top layer is mostly removed, a clean pipette
tip can be inserted from the top layer into the second layer. Light
agitation of the tip can be used to clear the interface from the
tip. The desired layer can then be drawn up in the pipette. The
interfaces above and below the desired layer should not be drawn up
with the desired layer to avoid layer contamination. In yet another
aspect, a plastic tube is side punctured one or more times using a
needle, such as a 21 to 16 gauge needle, to puncture the tube at
the desired phase. The desired phase is pulled from the tube
volume. In each of these aspects, if the analyte of interest is in
a phase, the interfaces above and below the phase should not be
disturbed to avoid layer contamination. Similarly, if the analyte
of interest is in an interface, the phases above and below the
interface should not be disturbed to avoid layer contamination.
[0119] The MPS system can be designed to separate living cells or
other biologically sensitive biological systems, e.g., ova, worms,
parasites, insects, protozoa, arachnids, fungi, annelids, and
arthropods, without harm to the living system. An MPS system
designed to separate biologically sensitive systems may include
without limitation GRAS polymers, biocompatible salts, isotonicity
with blood plasma, reagents that maintain the viability of
separated cells or are used to probe the activity of separated
cells, lytic or non-lytic reagents, biocompatible buffer conditions
that maintain a physiological pH, and dyes or stains to label
analytes and enhance detection.
[0120] In one aspect of one or more embodiments, analytes of
interest can be tagged to alter their density. Analyte tags can be
used for example to assist in the separation of analytes having the
same or substantially same density as other objects or impurities
in the sample. The sample components can be separated according to
their densities when the analytes of interest, other objects or
impurities are tagged. For example, the analyte of interest can
have a binding affinity for the tag molecule such that the analyte
of interest and the tag molecule link to form a tag
molecule-analyte adduct. The tag molecule-analyte adduct forms when
the tag molecule covalently bonds, non-covalently bonds,
hybridizes, complexes, or conjugates with the analyte of interest.
Tags can include beads of higher or lower density to that of the
analyte that have been treated to provide a chemical surface that
can interact with the analyte. Polymer beads, metal beads or glass
beads having a range of densities can be chemically modified, for
example with one of biotin or avidin, antibodies and the like. The
complementary surface on the analyte of interest, will cause the
analyte to bind to the bead surface. A non-limiting example of
using an adduct to separate analytes of like density includes the
use of antibodies, conjugated to gold colloids, that are specific
for cell markers expressed by a single desired cell type within a
range of densities that capture a variety of cell types such as,
e.g., separating CD4+ T cells from a mixture of leukocytes of
equivalent density. Other non-limiting examples of using an adduct
to separate analytes of like density include separating
antigen-presenting cells for antigen analysis for vaccine
development, differentiating hematopoietic stem cells CD34+ and
CD38-, differentiating cultured stem cells from feeder cells in
stem cell therapies, and distinguishing pathogenic and
non-pathogenic particles based on surface markers indicative of
virulence (e.g., distinguishing pathogenic E. coli from
non-pathogenic strains based on the presence or absence of the
surface marker intimin). Tags can also include larger biomolecules
that have affinity for the analyte of interest. Upon binding, the
density of the analyte is altered.
[0121] Biological carriers can also be separated from each other,
or from other objects or impurities in the sample, using the
methods disclosed herein. For example, complex biological carriers
that contain a variety of components can be screened for the
presence or absence of certain components that are useful for
health and public safety. MPS screening can be used to examine food
for adulteration, confirm presence or absence of heavy metals in
foods, screen bodily fluids such as blood, feces, saliva and urine
for the presence of parasites or infection.
[0122] In one aspect, the disclosed methods can be used to analyze
or separate cell lysates. Cells can be lysed while in the MPS, or
they can be lysed and later introduced to the MPS. Cells can be
lysed using methods known in the art. For example, cells can be
lysed in the MPS by treating the MPS with additives or agents, such
as hypotonic buffers that disrupt cell membranes or cause cells to
swell and burst. Lysozyme can be used to digest cell walls to free
and subsequently separate cellular components of bacteria and yeast
based on their densities using a MPS. Cells can also be lysed using
other methods, including but not limited to, the addition of lytic
agents including some surfactants, manual grinding, liquid
homogenization, sonication, and freeze/thaw methods. Manual
grinding using mortar and pestle is commonly used to disrupt plant
cells. Plant tissue can be frozen in, e.g., liquid nitrogen, and
then crushed and ground using a mortar and pestle to lyse cells.
Other mechanical methods of mechanical disruption include blenders.
Blenders can be used, for example, to grind and disperse large
amounts of tissue such as muscle and organs. For smaller samples
and cultured cells, liquid-based homogenization can be used to lyse
cells. Examples of liquid homogenizers include Dounce homogenizers,
Potter-Elvehjem homogenizers, and French presses. Sonication, which
uses high frequency sound waves to agitate and lyse cells, can be
used to lyse small volumes of cells, bacteria, and thinly sliced
tissue samples. The freeze/thaw method can be used to disrupt many
types of cells, including mammalian and bacterial cells.
[0123] In other embodiments, the disclosed methods can be used to
distinguish normal cells from parasitized, infected, rare, and
tumor cells. For example, cells infected by viruses or parasites
have densities that are either the same or different than normal
cells, depending on the type of virus or parasite with which the
cell is infected. Samples containing infected cells having
different densities than normal cells can be separated from other
objects or impurities in the sample using the disclosed methods to
separate infected cells from normal cells. For example, cells
infected with malaria lose density as the infection progresses.
Thus, in some embodiments, an aqueous multi-phase polymer system
comprising two or more polymer aqueous solutions or phases is used
to isolate malaria-infected blood cells.
Examples of Density-Based Separation Using Multi-Phase Systems
Example 1
[0124] The layers of an aqueous multiphase system are ordered
according to density, and therefore can be used to achieve
density-based separation
[0125] 23 polymers and 11 surfactants were investigated for their
ability to promote phase separation in aqueous solutions. These
reagents were used as prepared by the commercial manufacturer
without further purification, and the molecular weights of the
polymers used were polydisperse. For example, the polydispersity
index of the poly(2-ethyl-2-oxazoline) species used for these
assays is .about.3-4 for a molecular weight of 200 kDa. D.sub.2O as
a co-solvent or salts were added to affect phase density.
[0126] Two-component mixtures of the polymers and surfactants were
prepared and selected for screening by vortexing equal volumes of
stock solutions at high concentrations for 30 seconds to ensure
complete mixing. The mixtures were then centrifuged for five
minutes at 2000 g. A visually discernible interface characterized
those mixtures that separated into discrete phases, while miscible
solutions resulted in a homogenous solution. A number of mixtures
resulted in the formation of either a gel or a precipitate, which
were considered distinct from those mixtures that generated two
liquid phases. Dispersed surfactant micelles also were not
considered to be a unique phase in the context of this
experiment.
[0127] An ordering system based on this empirical miscibility data
and consistent within the set of reagents used in this experiment
was developed. A series of miscibility profiles was generated for
each reagent by assigning a 34-component vector describing the
results from all two-component mixtures that include the reagent.
The vector has values `0` for mixtures that resulted in homogeneous
solutions (miscible), `1` for mixtures that resulted in a
precipitate or a gel (incompatible), and `2` for mixtures that
resulted in phase separation (immiscible).
[0128] The miscibility profiles of N reagents and clusters of
reagents can be compared by analyzing the magnitudes of each vector
and the distances between vectors in N-dimensional space: a small
distance between vectors indicates similar miscibility profiles.
Referring to FIG. 12, reagents were ordered in the matrix according
to this vector analysis.
[0129] Using this approach to ordering, several patterns were
identified based on similarities in miscibility in two-component
mixtures: neutral, branched polysaccharides (numbers 3 and 4),
acrylic acids (4 and 5), cationic species (10, 12, and 13),
hydrophobic species incorporating ethylene oxide units (14-18), and
anionic species (21, 22, 24, 26-29) are clustered by patterns of
miscibility using our analysis.
Example 2
[0130] The aqueous multi-phase polymer systems used to isolate
malaria-infected blood cells are exemplified by the poly(ethylene
glycol)-dextran, dextran-Ficoll systems, and systems containing
poly(2-ethyl-2-oxazoline), poly(vinyl alcohol), Ficoll,
poly(ethylene glycol), dextran poly(2-vinylpyridine-N-oxide),
cellulose derivatives, polyvinylpyrrolidone, and combinations
thereof.
[0131] Malaria is traditionally diagnosed by combining the
observation of physical symptoms (i.e., a sustained fever) with
confirmation of parasitemia by microscopy. Malaria, however, is
most prevalent in rural regions of developing nations where limited
access to trained physicians and suitable clinical laboratory
equipment hinders the traditional diagnostic approach. Availability
of a low-cost, point-of-care test for diagnosing malaria would
significantly reduce the burden of this disease in the developing
world.
[0132] The method of separating analytes in a sample is
demonstrated in a specific embodiment in which an aqueous tri-phase
polymer system is used to separate malaria-infected erythrocytes in
whole blood from healthy blood cells based on their different
densities. This AMPS comprises a mixture of poly(vinyl alcohol)
(PVA), poly(ethylene glycol) PEG, and dextran. The density barriers
at the polymer/polymer interface of the triphasic PVA/PEG/dextran
system were established using the eggbeater centrifuge and were
designed to isolate white blood cells at the PVA/PEG interface,
infected red blood cells at the PEG/dextran interface (, and
healthy red blood cells in the pellet from whole blood. The healthy
red blood cells have a density of .rho..apprxeq.1.100 g/cm.sup.3
and the red blood cells infected with malaria parasites have a
density of .rho..apprxeq.1.080 g/cm.sup.3. Each polymer/polymer
interface was designed to isolate a specific component of blood:
the 10 wt/vol. PVA (.rho.=1.022 g/cm.sup.3)/40 wt % PEG
(.rho.=1.067 g/cm.sup.3) interface models the capture of white
blood cells, the 40 wt % PEG/30 wt % dextran (.rho.=1.101
g/cm.sup.3) interface models the capture of malaria-infected red
blood cells, and all healthy red blood cells are expected to
sediment to the distal end of the tubing. Thus, the blood cells can
be separated based on their small difference of densities, which
provides a quick and easy identification method of malaria
infection.
[0133] The PVA/PEG/dextran system described above is used herein to
demonstrate the concept of separating malaria-infected blood cells
from the healthy blood cells using density standards which
represent the density of the malaria-infected blood cells and of
the healthy blood cells. In another embodiment, the
malaria-infected blood cells can be separated from the healthy
blood cells using an ATPS comprising dextran/Ficoll and/or
PVA/dextran/Ficoll. The use of any one of the AMPSs disclosed
herein to separate malaria-infected blood cells from the healthy
blood cells is contemplated.
[0134] Referring to FIG. 6, an image of a separation achieved using
a two-phase aqueous polymer system comprising layers of dextran and
Ficoll to separate a mixture of red blood cells into two
populations by centrifugation is shown. The result of this
separation is shown in FIG. 6. Red blood cells infected with the
malaria parasite Plasmodium falciparum (6% of cell population) were
concentrated at the interface between polymer layers (see FIG. 6)
and healthy red blood cells (94% of cell population) were
sedimented through both polymer layers (see FIG. 6). Applicants
designed the polymer system such that their densities (1.079
g/cm.sup.3 and 1.085 g/cm.sup.3, respectively) would selectively
facilitate the separation of the two cell types in the sample. Each
layer was isotonic with respect to human plasma (300 mOsm/kg) and
at physiological pH (7.40).
[0135] FIG. 7 shows parasitized from healthy erythrocytes. This
figure shows separation of erythrocytes in which a two-phase AMPS
was used as a malaria diagnostic. Panel A of FIG. 7 shows images of
capillaries that were used to compare the separation patterns of
ring stage parasitized erythrocytes and healthy erythrocytes. The
presence of a red band at the interface indicated the capture of
parasitized cells, thus diagnosing malaria infection. Also
referring to FIG. 7, Panel B shows an image from a micrograph of
cells were isolated from the AMPS interface after smearing and
staining. This panel depicted the near complete enrichment of
parasitized erythrocytes due to density-based separation by
AMPS.
Example 3
[0136] The following example demonstrates that living cells can be
separated from a biological carrier, without affecting the
viability of the living cells. The analyte can be extracted or
removed for further investigation, e.g., culturing, quantifying the
results, etc. FIGS. 3A-C show smears from cultures of P.
falciparum-infected erythrocytes after separation from lymphocytes
from human whole blood.
[0137] The cultures were separated using a two-phase PEG/Ficoll
system. The system was prepared using dextran 500K at 20% (w/v),
Ficoll 400K at 23.5% (w/v) with 3 mM Na2 EDTA at a pH of 7.40. NaCl
was added to achieve an osmolality of 295 mOsm+/-15. The two phase
system was formed and the phases were separated. The phases were
then introduced by pipette into a hematocrit tube. 7 microliters of
the bottom phase was followed by 7 microliters of the top phase,
followed by 7 microliters of the sample (RBCs or parasitized RBCs).
The samples were washed with buffer once before introduction to the
system. The system was sealed and spun in a hematocrit centrifuge
at 13,000 g for 2 minutes after which a clear difference was
observable.
[0138] The erythrocytes were studied for two days after infection.
FIG. 3(A) is an image of the infected erythrocytes on the same day
as separation. At this stage, the infection is predominantly at the
ring stage and early trophozoites. FIG. 3(B) shows the progress of
the infection one day after separation (predominantly late
trophozoites and schizonts). Referring to FIG. 3(C), two-days after
separation predominantly shows rings and early trophozoites in
newly infected cells.
Example 4
[0139] Separation of small samples can be visualized without
disturbing the density of the system using dyes or
fluorophores.
[0140] Referring to FIG. 2, crystal violet was used to visualize
small amounts of E. coli filtered out of the blood. The E. coli
were prepared using standard methods. E. coli (laboratory strain
BL21(DE3)pLysS) were incubated overnight at 37.degree. C. in
Lauria-Bertani nutrient broth supplemented with 34 .mu.g/mL of
chloramphenicol. A PEG/Ficoll system with density steps at
approximately 1.028 and 1.096 g/mL separated the denser E. coli
from the human blood components. The system was prepared with PEG
20K at 15% mixed with Ficoll 70K at roughly 30%. The solutions were
mixed and the emulsion was adjusted to pH 7.40 and NaCl was added
to bring osmolality to 295 mOsm+/-15. Once the E. coli were
introduced to the system, the system was centrifuged at 13,000 g
for 4 minutes.
[0141] The crystal violet dye allowed a smaller concentration of E.
coli to be detected by eye than un-stained bacteria (compare to
FIG. 1). To achieve the separation shown in FIG. 2, the two phase
PEG/Ficoll system was premixed and separated out. Then, 5
microliters of the bottom phase, 5 microliters from the top phase,
5 microliters of crystal violet dye, and 5 microliters of sample
blood (E. coli doped or control) were added to a tube which was
sealed and centrifuged to arrive at the separation depicted in FIG.
2.
Example 5
[0142] This example demonstrates the separations of components of a
biological fluid.
[0143] Referring to FIG. 4, a two-phase PEOZ/Ficoll system was used
to separate cells and plasma from whole blood. A capillary was used
to separate components of whole blood by density (center). An
illustration of the density step is also shown (left). The system
was prepared with PEOZ 50K at 20% with Ficoll 400K at 20%. pH 7.35
and osmolality around 350 mOsm. (In other embodiments, the system
is prepared with a PEG 20K 15%/and Ficoll 70K at 12.5% at a pH of
7.40.) 30 microliters of the mixed two-phase system was added to a
capillary tube that was capped and spun for 15 minutes to achieve
phase separation. The tube was uncapped to add 20 microliters of
blood, re-capped, and spun for 14 minutes at about 2000 g in a
swinging bucket table top centrifuge. The tubes were cut and
sections were removed to make smears for slides. Brightfield
microscope images showed smears from isolated sections of the
capillary tube dyed with two-part stain (FIG. 4, right) in which
erythrocytes, leukocytes, and cell debris were shown to separate
and migrate to different parts of the MPS based on each component's
density.
Example 6
[0144] The methods disclosed here combine the portability and
simplicity of the soft centrifuge with aqueous multiphase density
barriers generated from polymers that exhibit limited interaction.
Aqueous polymers that exhibit limited interaction have numerous
advantages over discontinuous density gradients for field use: they
are easily prepared, owing to the nature of their clear boundaries;
they are stable, and thus amenable to long term storage; and they
are versatile in that can be altered (composition and/or density)
to suit the application.
[0145] Moreover, the soft centrifugation assay requires no more
than 10 .mu.L of whole blood (easily obtained from a single
fingerstick), and rapidly separates blood components. In some
embodiments, the soft centrifugation takes 10 minutes or less.
Examples of Separating Tagged Analytes Using Multi-Phase
Systems
[0146] The density of analytes can be modified using tag molecules.
Tag molecules change the density of tagged species by either
increasing or decreasing the density of the tagged species.
Non-limiting examples of particles that can be used to tag analyte
include gold colloids, which increase the density of a tagged
species, and hollow glass spheres, which decrease the density of a
tagged species. Particles of varying sizes can be used depending on
the magnitude of the desired density shift. Sizes ranging from 5
nm-50 .mu.m are contemplated.
[0147] Tag molecules can be formed a variety of ways. Non-limiting
examples for forming tag molecule include conjugating a dense
particle directly to a ligand capable of binding the surface of an
analyte of interest (e.g., labeling an antibody to CD4 with a gold
colloid), and introducing to the sample a particle capable of
binding a ligand without interfering with the ligand's ability to
bind to the analyte of interest (e.g., a streptavidin-coated gold
colloid capable of binding a biotinylated antibody to CD4).
[0148] The tag molecule-analyte adducts can be formed by several
methods. The formation of the tag molecule-analyte adduct can be
performed in a stepwise manner (e.g., incubating the dense particle
with the ligand to form a complex (tag molecule) that is incubated
with the analyte of interest before introduction to the MPS, or
incubating the analyte of interest with the dense particle and
ligand to form a tag molecule-analyte adduct before introduction to
the MPS). In another aspect, the tag molecule-analyte adduct is
formed when the analyte of interest is introduced to a MPS in which
the tag molecule is admixed with one or more phases.
[0149] In one or more embodiments, phases comprising phase
components such as salts or surfactants that attract or repel
tagged analytes of interest cause the tagged analytes to
preferentially accumulate in one of the phase or at an interface in
the MPS by passing through one or more phases sequentially, while
other impurities or components in the sample preferentially
accumulate in another phase or at another interface of the MPS,
thus separating the tagged analytes from the rest of the sample. In
one aspect, this embodiment can be used to separate analytes of
interest from samples comprising impurities with densities similar
to that of the analyte of interest. In this aspect, the analyte of
interest is linked to the tag molecule to form a tag
molecule-analyte adduct having a density that is different than the
density of the impurity, other objects in the sample, any untagged
analyte, or combinations thereof. The MPS has one or more phases or
phase components to which the analyte, impurity, other objects,
and/or untagged analyte preferentially migrate. The MPS has one or
more phase components consisting of surfactant, polymer, or both.
The tag molecule-analyte adduct can then be separated from the
impurity, other objects, or untagged analyte by adding the sample
to the MPS. Because the tag molecule-analyte adduct has a density
that is different than the density of the impurities, other
objects, or untagged analyte the tag molecule-analyte adduct and
impurities separate and move to different locations in the MPS by
passing through one or more phases sequentially.
[0150] In some aspects, the disclosed methods for analyzing and
separating samples can be used to determine whether a sample
contains cells infected by a particular infectious agent using tag
molecules that are attracted to or repelled by phases containing
certain phase components. For example, cells infected by viruses
produce viral proteins, some of which migrate to the cell's outer
membrane where the proteins form a complex that identifies the cell
as infected. Tag molecules that preferentially link to membrane
proteins characteristic of a certain virus can be introduced to a
sample that may or may not contain cells infected by that virus.
For example, a sample that may contain a certain virus can be
contacted with a tag molecule specific for a particular viral
protein. The tag molecule changes the density of the tagged virus
such that it has a different density than other objects or
impurities in the sample. If the virus is present in a sample, the
viruses are tagged with the tag molecule. When the tagged viruses
are introduced to the MPS, the tagged virus preferentially migrates
to a particular phase or phases based on its density, passing
through one or more phases sequentially until it eventually comes
to rest in a phase or at an interface with the same density as the
tagged virus. The phase, phases, or interface containing the tagged
virus can optionally be separated from the system to further
separate or analyze the tagged virus.
[0151] Similarly, in another embodiment, the disclosed methods can
be used to determine whether a sample contains rare cells such as
activated lymphocytes, stem cells, fetal cells and tumor cells. In
still another embodiment, the disclosed methods can be used to
distinguish benign tumor cells from cancer cells. Cells use
signaling proteins to respond to their environment. For example,
many tumor cells, including cancer cells, have membrane proteins
that are characteristic of the type of tumor or cancer that has
affected the cell. In these embodiments, a tag molecule capable of
linking to the membrane protein can be selected provided the tag
molecule gives the cell affected by tumor or cancer a different
density compared to normal cells. The tag molecule is also selected
based on its ability to link to the membrane protein on the
infected cell. The tag molecule can be linked to the membrane
protein by covalent bonding, non-covalent bonding, hybridization,
electrostatic interactions, complexing, and conjugation to form a
tag molecule-analyte adduct. The sample containing the tag
molecule-analyte adduct can be added to the MPS, and because the
density of the tag molecule-analyte adduct (i.e., tagged, infected
cell) is different than the density of normal cells, infected cells
that have been tagged and normal cells will move to different
locations in the MPS passing through one or more phases
sequentially. Similarly, tag molecules specific to membrane
proteins characteristic of other rare cells such as stem cells,
activated lymphocytes, and fetal cells can be used according to the
methods disclosed herein to separate tagged rare cells from normal
cells.
Example 7
[0152] Samples containing cells of interest that have the same
density as other cells in the sample can be tagged to separate the
cell type of interest from the rest of the sample using
density-based separation in a MPS.
[0153] CD3-/CD4+ cells are separated from a co-culture of T cell
lymphocytes containing CD3+/CD4- and CD3-/CD4+ cells. The cells are
cultured using standard methods. The density of each cell type are
determined using a MPS, and are found to be identical (.rho.=1.055
g/cm.sup.3).
[0154] 1 mg/mL streptavidin-coated 50 nm gold nanoparticles are
incubated with 100 .mu.g/mL biotinylated antibody to CD4 for 2
hours in isotonic PBS buffer to form an immunocomplex solution.
[0155] The cells are harvested from the culture by centrifugation
at 4000 g for 2 minutes. The supernatant is removed and the cells
are resuspended in an amount of cell culture medium sufficient to
double the cell concentration. The cells are diluted 1:1 with the
immunocomplex solution and incubated for 1 hour.
[0156] The cells are introduced to a two-phase MPS and sedimented
by centrifugation at 4000 g for 10 minutes. The MPS separates the
cells into three populations: cell debris (top of system);
CD3+/CD4- T cells (interface between phases); and tagged CD3-/CD4+
cells (bottom of the system). The tagged CD3-/CD4+ cells are
isolated by pipette and resuspended in cell culture medium for
further analysis or culturing.
[0157] Upon review of the description and embodiments of the
present invention, those skilled in the art will understand that
modifications and equivalent substitutions may be performed in
carrying out the invention without departing from the essence of
the invention. Thus, the invention is not meant to be limiting by
the embodiments described explicitly above, and is limited only by
the claims which follow.
* * * * *